CHARACTERIZATION OF LACTOBACILLUS ... - Semantic Scholar

Brazilian Journal of Microbiology (2011) 42: 158-171 ISSN 1517-8382

CHARACTERIZATION OF LACTOBACILLUS FROM ALGERIAN GOAT’S MILK BASED ON PHENOTYPIC, 16S rDNA SEQUENCING AND THEIR TECHNOLOGICAL PROPERTIES Ahmed Marroki1 *; Manuel Zúñiga2; Mabrouk Kihal3; Gaspar Pérez- Martínez2 1

Laboratory of Applied Microbiology, Department of Biotechnology (IGMO), University of Oran Es-Sénia, Oran, 31100 ,

Algeria; 2Department of Biotechnology, Instituto de Agroquímica y Tecnología de los Alimentos (C.S.I.C), polígono de la Coma s/n, Burjassot (Valencia), Spain; 3Laboratory of Applied Microbiology, Department of Biology, University of Oran Es-Sénia, Oran, Algeria. Submitted: February 18, 2010; Returned to authors for corrections: February 24, 2010; Approved: August 01, 2010.

ABSTRACT Nineteen strains of Lactobacillus isolated from goat’s milk from farms in north-west of Algeria were characterized. Isolates were identified by phenotypic, physiological and genotypic methods and some of their important technological properties were studied. Phenotypic characterization was carried out by studying physiological, morphological characteristics and carbohydrate fermentation patterns using API 50 CHL system. Isolates were also characterized by partial 16S rDNA sequencing. Results obtained with phenotypic methods were correlated with the genotypic characterization and 13 isolates were identified as L. plantarum, two isolates as L. rhamnosus and one isolate as L. fermentum. Three isolates identified as L. plantarum by phenotypic characterization were found to be L. pentosus by the genotypic method. A large diversity in technological properties (acid production in skim milk, exopolysaccharide production, aminopeptidase activity, antibacterial activity and antibiotic susceptibility) was observed. Based on these results, two strains of L. plantarum (LbMS16 and LbMS21) and one strain of L. rhamnosus (LbMF25) have been tentatively selected for use as starter cultures in the manufacture of artisanal fermented dairy products in Algeria. Key words: Lactic acid bacteria; Lactobacillus; identification; goat’s milk; technological properties; Algeria.

INTRODUCTION

the considerable variations in biochemical attributes between strains currently considered to belong to the same species. In

The identification of lactobacilli has been based mainly on

fact, some species are not readily distinguishable in terms of

fermentation of carbohydrates, morphology, and Gram staining

phenotypic characteristics (12). In recent years, the taxonomy

and these methods are still being used. However, the

has changed considerably with the increasing knowledge of the

characterization of some Lactobacillus to species level by

genomic structure and phylogenetic relationships between

biochemical methods alone is not reliable (27, 40), because of

Lactobacillus spp. (27, 43, 47). This novel taxonomy based on

*Corresponding Author. Mailing address: Laboratory of Applied Microbiology, Department of Biotechnology (IGMO), University of Oran Es-Sénia, Oran, 31100, Algeria.; Tel: +213 772 142 345 Fax: + 213 41 58 19 41.; E-mail address: [email protected]

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Marroki, A. et al.

Characterization of Lactobacillus and their technological properties

MATERIAL AND METHODS

DNA analysis offers a variety of advantages over other more conventional typing procedures, such as the stability of the genomic DNA analysis, the capacity to discriminate bacteria at

Milk samples

the strain level, and the amenability to automation and

Five samples of goat’s milk collected from farms located

statistical analysis (21). These methods have been employed

in the region north-west of Algeria were used in this study. The

for differentiation or identification and typing of different

samples were collected aseptically in sterile bottles kept in an

species of Lactobacillus. The species of lactobacilli most

ice-box, and transported immediately to the laboratory.

commonly found in milk, and dairy product, especially in goat’s milk are L. plantarum, L. rhamnosus, L. casei or L.

Phenotypic characterization

paracasei. For this reason, the selection of Lactobacillus

One milliliter of each milk sample was homogenized with

strains from goat’s milk has been considered in the search for

9 ml of sterile Ringer’s solution 1:4 and mixed thoroughly for

new industrially important cultures, in order to select those

60s. Serial dilutions were made and aliquots (100 µl) of each

with the highest potential for industrial applications. In Algeria,

dilution were streaked on MRS agar (Oxoid, UK) (17). The

goat’s milk plays a vital role in human consumption, most

MRS plates were incubated at 30 °C and 45°C for 24 to 48h

being consumed by the rural community, while little is

under anaerobic conditions (Anaerogen, Oxoid). Ten colonies

available on the market (5). Algerian people make various

from plates corresponding to the highest dilutions were

fermented dairy products using goat’s milk. The transformation

randomly selected and purified by subculturing. Gram-positive,

of goat’s milk into traditional Algerian dairy products, such as

catalase negative cultures were stored at -80 °C in MRS

El – Klila, a traditional cheese which is popular in the country

supplemented with 20% glycerol. Isolates were phenotypically

side and is made from unpasteurised cow or goat surplus milk

assigned to the genus level on the basis of: cell morphology,

(7), Jben (local traditional fresh cheese), Raïb, and Lben (local

Gram-positive and catalase-negative, according to the methods

traditional fermented milks), is achieved through spontaneous

and criteria described by Sharpe (42) and Kandler and Weiss

fermentation without the addition of any selected starter. Such

(26); CO2 production from glucose in MRS broth containing

products generally present irregular sensorial qualities. The aim

inverted Durham tubes (32); hydrolysis of arginine, growth at

of the present study was to characterize Lactobacillus isolated

15 °C and 45 °C, tolerance to 20, 40, 65 g L-1 NaCl. The acid

from goat’s milk from north-west of Algeria, using

production from carbohydrates (fructose, glucose, mannitol,

physiological, phenotypic and genotypic methods. There are no

lactose, mannose, rhamnose, glycerol, arabinose, sorbose,

previous reports concerning the genetic identification of

dulcitol, amygdalin, melibiose, melezitose, starch, tagatose,

Lactobacillus or studies that combined the phenotypic and the

arabitol, ribose, maltose, galactose, and xylose) was evaluated

genotypic identification of Lactobacillus isolated from goat’s

by using a miniaturized assay in microtiter plates, as described

milk in Algeria. Additionally, in order to select adequate

by Jayne-Williams (25). Ability to ferment carbohydrate

strains susceptible to be used as starter cultures for the

substrates was studied, using the API 50 CHL system

manufacture of artisanal fermented dairy products in Algeria,

(BioMérieux, Lyon, France), following the manufacturer

some important technological properties, including the capacity

recommendations.

of acidification/coagulation of skim milk, exopolysaccharide production,

aminopeptidase,

antibacterial

antibiotics resistance, were also studied.

activity,

and

DNA extraction and 16S rDNA sequencing Isolates were grown in MRS broth at 30 °C until OD of

159

Marroki, A. et al.

Characterization of Lactobacillus and their technological properties

1.6 - 1.8 at 600 nm. A 1.5 mL aliquot of each overnight culture

pH changes were measured with a pH meter (glass electrode,

was centrifuged at 10000 × g for 30 s at room temperature in

Crison, Spain) after 6, 12, and 24 h of incubation at 30°C.

order to pellet cells. Bacterial DNA was isolated by using the

Acidification activity was measured by following the change in

TM

(MoBio

the pH during time. Coagulation of milk was determined after

Laboratories Inc., Solana Beach, CA, USA), following the

24 h of incubation at 30°C. Screening of exopolysaccharide

instructions of the manufacturer.

(EPS) was carried out in ruthenium red milk plates, as

UltraClean

Microbial

DNA

isolation

Kit

The 5′ end variable region of the 16S rDNA was PCR-

described by Stingele et al. (44).

amplified with primers 27F (5′-AGAGTTTGATCCTGGCTC

Aminopeptidase activity of the strains was determined

AG-3′) and 558R (5′-GTATTCCGCGGCTG-3) or with the

using the synthetic substrates L-alanine ρ-nitroaniline (Ala-ρ-

primers 27F and 1525R (5′-AAGGAGGTGWTCCARCCG

NA) (Sigma,USA), and L-leucine ρ-nitroaniline (Leu-ρ-NA)

CA-3′) using a total volume of 50 µl containing 50 ng of DNA,

(Sigma,USA) as described by Zotta et al. (51). Stationary

25 pmol of each primer, 1.6 mM of dNTPs, 2 mM MgCl2 and

phase cells grown overnight in MRS broth were harvested by

1U of Taq DNA polymerase (Biotools Lab, Spain), using the

centrifugation at 10000 × g for 5 min, washed twice with sterile

reaction buffer supplied by the manufacturer.

50 mM potassium phosphate buffer, pH 7.0, and re-suspended

Amplifications were carried out in a Thermal Cycler

in the same buffer to obtain cell suspensions of (A

650

= 1.0).

(PTC-100 Peltier Themal Cycler, MJR), using the following

Aminopeptidase activity was measured, according to Macedo

program: for primers 27F and 558R, the 16S rDNA was

et al.(31). The assay mixture contained: 30µl of 20 mM

amplified as described by Linaje et al. (30); for the primers 27F

aminoacyl ρ-nitroanilide substrates dissolved in methanol, 195

and 1525R, the PCR mixtures were subjected to an initial

µL of 50 mM potassium phosphate buffer (pH 7.0), 95 µL of

denaturing step of 95 °C for 5min, followed by 30 PCR cycles

0.05% (w/v) sodium azide solution, and 75 µL of cell

(94 °C, 15s; 52 °C, 30s; 72 °C, 2min) and final cycle at 72 °C

suspension. After incubation at 30 °C for 1 to 4 h, the reaction

for 5min. The PCR products were subjected to gel

was stopped by the addition of 900 µL of 1% (v/v) acetic acid.

electrophoresis in 1% agarose gel, followed by staining with

The release of ρ-nitroaniline (ρ-NA) (Sigma, USA) was

ethidium bromide and visualization under UV light. A Lambda

measured spectrophotometrically (Hewlett Packaro, Diod

DNA (Biorad) digested with PstI ladder was used as a

Array Spectrophotometer,

molecular mass marker.

centrifugation of the mixture at 10000 × g for 5min. Data

Germany) at 410 nm after

Polymerase chain reaction products were purified by using

obtained were compared to a calibration curve prepared using

the GFX PCR DNA and a gel band purification Kit (General

ρ-NA (Sigma, USA) dilutions ranging from 0.1 to 20.0 mM.

Electric Healthcare, Spain), following the manufacturer’s

One unit of enzyme activity was defined as the amount of

instructions. DNA sequencing was carried out by the Central

enzyme required to release 1 µmol of ρ-NA min-1 under the

Service of Research Support of the University of Valencia

assay conditions.

(Spain),

by

using

the

dideoxynucleotide

DNA

chain

termination method.

Antibacterial activity of Lactobacillus strains was tested with the well diffusion method described by Linaje et al. (30). Cells were grown overnight in MRS broth. Cell-free

Technological properties Acidifying activity in skim milk was assayed as described

supernatants were obtained by centrifugation at 10000 × g for 10 min at 4 °C, adjusted to pH 6.5 with 1N NaOH, and then

by Psono et al. (35). Sterile skim milk samples (100 mL; 1.0%)

filtered

were inoculated with overnight cultures which had been

(Millipore). The supernatants were adjusted to pH 6.5, in order

previously activated by two successive transfers in milk. The

to eliminate the eventual antimicrobial activity linked to

through

0.22µm

Durapore

membrane

filters

160

Marroki, A. et al.

Characterization of Lactobacillus and their technological properties

RESULTS AND DISCUSSION

organic acids. A 100 µL aliquot of an overnight culture of the indicator bacteria Listeria monocytogenes CECT 932T, Bacillus cereus INRA AVZ 421, Staphylococcus aureus CECT 86T and Staphylococcus aureus UT 602, was mixed with 5 mL of soft

Phenotypic identification of isolates Isolated

strains

were

identified

based

on

their

agar (Trypticase Soya Broth for Bacillus cereus and

physiological and biochemical characteristics given by Kandler

Staphylococcus aureus and Brain Heart Infusion for Listeria

and Weiss (26), Dellaglio et al. (15) and Stiles and Holzapfel

monocytogenes, supplemented with 0.8% agar). Aliquots (50

(43), to the species level, and also by using API 50 CHL test

µl) of supernatant of overnight cultures were poured in the

strips (BioMérieux, Lyon, France) for confirmation of species

wells digged in the soft agar. After 24 h of incubation at 37 °C,

of selected strains.

inhibition zones were read. A clear zone of inhibition >1 mm

All isolates (19 Lactobacillus strains) were rod shaped

around a well was scored as positive. In order to check the

cells, Gram-positive, catalase-negative, non motile and

thermoresistance of the bacteriocins, cell free supernatant

facultative anaerobic bacteria. Isolates were classified as

samples were heated at 100°C for 10 min, prior the

belonging to the genus Lactobacillus. All isolates were able to

antibacterial assay. The proteinaceous nature of the inhibitory

grow at 15°C, 2%, and 4% NaCl. They were divided into two

activity was tested by the addition of 0.5 µg of proteinase K

preliminary groups (I and II), according to the results for CO2

(Roche Molecular Biochemicals) to the concentrated culture

production from glucose, and NH3 production from arginine:

supernatants (50 µL) distributed among the wells of the assay

Group I, facultatively heterofermentative, and arginine-

plates.

negative

lactobacilli

heterofermentative, Antibiotic susceptibility testing

and

(94.73%);

group

arginine-positive

II strain

strictly (one

Lactobacillus strain).

Susceptibility testing was based on the agar overlay disc

Table 1 shows the carbohydrate utilization patterns and

diffusion test described by Charteris et al. (10), as modified by

other physiological and biochemical characteristics of the

Aymerich et al. (4). Briefly, Lactobacillus strains were grown

lactobacilli isolates. The analysis of data compared with those

overnight in MRS broth at 30°C under anaerobic conditions

of the criteria given by several authors, resulted in four

(Anaerogen, Oxoid). Eight ml of MRS soft agar kept at 50°C

subgroups (A-D). Group I was subdivided into three subgroups

were inoculated with 200 µL of the grown culture. Petri dishes

(A-C) and group II, comprised only one subgroup (D). Isolates

containing 15 mL of MRS were overlaid with 8.2 mL of the

belonging to the four subgroups were able to ferment fructose,

inoculated MRS and allowed to solidify at room temperature.

glucose, mannitol, lactose, mannose, ribose, maltose, and

Antibiotic discs were placed onto the overlaid plates and all

galactose and unable to ferment xylose.

plates were incubated at 30°C for 24 h under anaerobic

•

Subgroup A was the largest one, with 11 strains (58%)

conditions. All isolates were screened for their susceptibility to

identified as L. plantarum. The strains were able to ferment

penicillin G (10 µg), ampicillin (10 µg), vancomycin (30 µg),

amygdalin, melibiose, melezitose, and arabitol, but unable to

tetracycline (30 µg), erythromycin (15 µg), kanamycin (30µg)

ferment rhamnose, glycerol, sorbose, dulcitol, and tagatose.

gentamicin (10 µg), and chloramphenicol (30 µg). Inhibition

However, variations in fermentation patterns were observed for

zones diameters of antibiotics were compared to those defined

some sugars: arabinose was fermented by 36%, starch by 18%,

by Charteris et al. (10) for lactobacilli.

sorbose by 9%, and dulcitol by 9% of the strains. Most of the

161

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Characterization of Lactobacillus and their technological properties

lactobacilli reported as being able to produce amylase are

(40%) to ferment glycerol resulted in their classification either

strictly homofermentative (26), although some L. plantarum

as L. plantarum or as L. pentosus (8, 50). Growth at 45°C was

have been reported as starch fermenting strains (33). Eighteen

observed for 40% of isolates.

percent of isolates from this subgroup were able to grow at

•

Subgroup C, included two isolates able to ferment

45°C. Some of the L. plantarum strains were capable of

rhamnose, arabinose, amygdalin, and melezitose. However,

growing at 45°C, in contrast to the characteristics given in

glycerol, sorbose, dulcitol, melibiose, starch, tagatose, and

Bergey’s Manual (26). Other studies have also reported the

arabitol were not fermented. All the isolates were able to grow

isolation of L. plantarum strains capable of growing at this

at 45°C and identified as L. rhamnosus.

temperature (20, 39). •

•

Subgroup D, with one isolate able to ferment

Subgroup B, comprised 5 strains (26%) identified as

rhamnose, arabinose amygdalin, melibiose and tagatose. In

belonging to L .plantarum/L. pentosus species. The isolates

contrast, this strain was incapable of fermenting glycerol,

only differed from those of subgroup A in the inability to

sorbose, dulcitol, melezitose, starch and arabitol. This isolate

ferment the starch and arabitol. The ability of some strains

was able to grow at 45°C and was classified as L. fermentum.

Table 1. Biochemical and physiological characteristics of Lactobacillus strains isolated from Algerian goat’s milk. Group Subgroup Number of isolates CO2 from glucose Arginine Hydrolysis Growth at 15°C 45°C Growth in 2 % NaCl 4 % NaCl 6.5 % NaCl Sugar fermentation Fructose Glucose Mannitol Lactose Mannose Rhamnose Glycerol Arabinose Sorbose Dulcitol amygdalin Melibiose Melezitose Starch Tagatose Arabitol Ribose Maltose Galactose Xylose

A 11 -

I B 5 -

C 2 -

II D 1 + +

+ 18

+ 40

+ +

+ +

+ + -

+ + -

+ + -

+ + -

+ + + + + 36 9 9 + + + 18 + + + + -

+ + + + + 40 + 20 20 + + + + + -

+ + + + + + + + + + + + -

+ + + + + + + + + + + + + -

+: Positive reaction; -: Negative reaction; 20: 20% of isolates showed a positive result; Subgroup A: LbMA9, LbMF13, LbMF33, LbMS4, LbMS9, LbMS14, LbMS16, LbMS20, LbMS21, LbMS24, LbMO16; Subgroup B: LbMO27, LbMO42, LbMS40, LbMT9, LbMT10; Subgroup C: LbMF24, LbMF25; Subgroup D: LbMA47.

162

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Characterization of Lactobacillus and their technological properties

C and D as L. rhamnosus and L. fermentum, respectively.

To establish the final phenotypic identification, all strains

A clear identification of species, especially within the

tested for their biochemical and physiological characteristics biochemical

genus Lactobacillus, based on phenotypic methods, such as

characterization, using API 50 CHL galleries (BioMérieux,

fermentation patterns, may sometimes be difficult, due to an

Lyon, France). The programme of identification (Cox and

increasing number of lactic acid bacteria species which vary on

Thomson, Biochemistry institute, Odense University) plus

a small number of biochemical traits (36). Commercially

database was used for the interpretation of the strains

available systems based on carbohydrate fermentation should

fermentation profiles (Table 2). The strains of the subgroups A

be combined with conventional phenotypic properties other

and B were classified as L. plantarum and those of subgroups

than carbohydrate fermentation or with genotypic techniques.

(Table

1),

were

submitted

to

further

Table 2. Fermentation of carbohydrates by Lactobacillus strains from Algerian goat’s milk, tested by the API 50 CHL system Groups

A

B

C

D

Isolates

LbMA9 LbMF13 LbMF33 LbMS4 LbMS9 LbMS20 LbMS14 LbMS16 LbMS24 LbMS21 LbMO16

LbMO27 LbMO42 LbMS40 LbMT9 LbMT10

LbMF24 LbMF25

LbMA47

Glycerol

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

+

-

-

-

L-Arabinose

+

+

+

+

-

-

-

-

-

-

-

-

+

+

+

+

+

+

+

L-Sorbose

-

-

-

-

-

-

-

-

-

-

-

+

-

-

-

-

-

-

-

Dulcitol Mathyl- DMannopyranoside Mathyle-DGlucopyrannoside N-Acetylglucosamine

-

-

-

-

-

-

-

-

-

-

-

+

-

-

-

-

-

-

-

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

-

-

+

+

+

+

+

+

+

-

-

+

-

+

-

+

+

-

-

+

+

+

+

+

+

+

+

+

+

+

+

+

+

-

+

+

+

+

+

+

+

Amygdalin

+

+

+

+

+

+

+

+

+

+

+

-

+

+

-

-

+

+

+

D-Melibiose

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

-

-

+

D-Melezitose

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

-

Starch

-

-

-

-

+

+

-

-

-

-

+

-

-

-

-

-

-

-

-

D-Tagatose

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

D-Arabitol

-

-

-

-

-

-

+

+

-

+

-

-

-

-

-

-

-

-

-

L-Arabitol + + + + + + + + + + + +: Positive reaction; -: Negative reaction. All isolates were able to ferment D-ribose, D-galactose, D-glucose,D-fructose,D-mannose, D-mannitol, D-sorbitol, arbutin, esculin, salicin, D-celibiose, Dmaltose, D-lactose, D-sucrose, D-trehalose, D-raffinose, gentibiose, D-turanose and gluconate. None fermented erythriol, D-arabinose, D-xylose, L-xylose, Dadonitol, methyl- -D-xylopyranoside, inositol, inulin, glycogen, xylitol, D-rhamnose, D-fucose, L-fucose, 2-ketogluconate and 5-ketogluconate.

-

-

-

-

-

-

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Marroki, A. et al.

Characterization of Lactobacillus and their technological properties

Genotypic identification of the isolates by 16S rDNA

acknowledged that L. plantarum and L. pentosus belong to the

sequencing

same 16S rRNA phylogenetic group and could only be

The 16S rDNA of the 11 strains of subgroup A and 5 isolates of the subgroup B was amplified by 27F and 558R

distinguished using phylogenetic analysis of sequences of the 16S-23S large spacer region (22).

primers, as reported by Acedo-Félix and Pérez-Martinez (1).

The comparative evaluation of phenotypic and genotypic

By partial sequencing of 16S rDNA all strains belonging to the

results confirmed that the phenotypic test, in spite of giving

subgroup A were classified as L. plantarum. Of the 5 isolates

information on the biochemical and metabolic traits of LAB,

forming subgroup B, 60% were identified as L. pentosus,

are not reliable enough for the identification of these

(LbMS40, LbMT9 and LbMT10) and 40% as L. plantarum

microorganisms, although it is a useful tool for presumptive

(LbMO42, and LbMO27). The 16S rDNA of two isolates of

classification. Lactobacillus UFV H2B20, a probiotic strain,

subgroup C were amplified with primers 27F and 1525R and

for example, which was first identified as L. acidophilus based

identified as L. rhamnosus (LbMF24 and LbMF25). The 16S

on its sugar fermentation profile (38), was afterwards classified

rDNA of the isolate of subgroup D was amplified by the

as L. delbrueckii using molecular methods (16). One major

primers used for subgroup C and identified as L. fermentum

reason for the mismatch between phenotypic and genotypic

(LbMA 47).

data might be ascribed to loosing or acquiring plasmids, which

Results of the PCR assay correlated with those obtained

leads to metabolite inconsistencies, as some carbohydrate

using the API 50 CHL system for 13 isolates identified as L.

fermentation capacities are plasmid encoded (2). Genotypic

plantarum, 2 isolates as L. rhamnosus, and one isolate as L.

techniques are doubtlessly rapid and accurate tools for the

fermentum. However, three isolates identified as L. plantarum

identification of LAB. The advantages of genotyping include

by the API system were found to be L. pentosus by sequencing

the stability of genomic DNA, its composition being

16S rDNA (Table 3). This result is not surprising, given that

independent of cultural conditions or preparation methods, and

the two species themselves have very similar 16S rDNA

amenability to automation and statistical data analysis (21).

sequences that differ only by 2pb (19). In fact, it is widely Table 3. Phenotypic and genotypic identification of Lactobacillus isolated from Algerian goat’s milk. Phenotypic dentification

Isolates

Genotypic identification (16S rDNA)

Most similar sequence (Acc. Nº)

Lactobacillus plantarum L. plantarum L. plantarum

LbMA9, LbMO16, LbMO27 LbMF13 LbMF33 LbMS4, LbMS9, LbMS14, LbMS16, LbMS20, LbMS21,LbMS24 LbMO42 LbMS40, LbMT9 LbMT10 LbMF24,LbMF25 LbMA47

Lactobacillus plantarum L. plantarum L. plantarum

AB362768.1 EF185922.1 AB362758.1

L. plantarum

EU257480.1

L. plantarum L. pentosus L. pentosus L. rhamnosus L. fermentum

AB362625.1 AB362758.1 AB362712.1 AB008211.1 AB362626.1

L. plantarum L. plantarum L. plantarum L. plantarum L. rhamnosus L. fermentum

Technological characteristics of strains

skim milk are shown in Table 4. Milk pH after 24 h of

Results on acidifying activity of Lactobacillus strains

incubation varied between 4.40 and 5.54 for all cultures. All

isolated from goat’s milk, after 6, 12 and 24 h of growth in

Lactobacillus isolates tested reduced the pH of milk to 6.43

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Marroki, A. et al.

Characterization of Lactobacillus and their technological properties

(6.12 - 6.43) after 6 h of growth, except for strains

species (49).

LbMA47,LbMS21 and LbMS16 which, respectively, were able

L. pentosus and L. rhamnosus strains showed a more

to decrease milk pH to values 5.31, 5.78 and 5.97. After 12 h,

homogenous acidifying behaviour. Strains of the first species

the pH values ranged from 5.24 (LbMS21) to 5.94 (LbMT10),

had a medium acidifying capacity with pH ranging between

while after 24 h, 47.37% of the strains had lowered milk pH to

0.43 and 0.58 pH units, after 6 h of incubation and achieved

4.40 - 4.96. According to their ability to reduce the pH more or

1.18-1.30 pH units until 24 h. Furthermore, the two L.

less rapidly, three clusters of L. plantarum isolates were

rhamnosus strains showed a fast acidifying activity, with

observed. (i) Slow acidifying strains (cluster I, 23% of L.

values of pH 0.63 and of 0.69 pH units after 6 h. After 24 h of

plantarum strains) showed a slow rate of acidifying ability

incubation the pH reached values of 1.87 and 1.96 pH units.

during the first 6 h of incubation ( pH ranging between 0.27-

L. fermentum strain showed the lowest rate of acidifying: pH

0.40 pH units). These L. plantarum strains lowered the

pH

0.25 pH units after 6 h and resulted in pH 1.35 pH units until

values between 0.86-1.01 pH units and 1.33-1.60 pH units after

24 h of incubation. Generally, L. plantarum strains produce

12 h and 24 h of incubation, respectively. (ii) Medium

acid more rapidly, when compared to other lactobacilli (41,

acidifying L. plantarum strains (cluster II) showed a faster rate

14). The fast acidifying strains (mostly L. plantarum and L.

of acidifying ability after 6 h of incubation, with a pH ranging

rhamnosus isolates) should be selected as part of a starter

between 0.50 and 0.57 pH units. In this cluster, two subgroups

preparation.

were observed with different behaviours in their acidifying

Seven isolates of L. plantarum strains and two L.

capacity. The first subgroup A (31% of L. plantarum strains) in

rhamnosus were able to coagulate skim milk, when inoculated

which the

pH values was achieved, respectively, 0.78-0.98

in skim milk after 24 h at 30 °C (Table 4). None of the L.

and 1.16-1.74 pH units, after 12 h and 24 h of incubation. On

pentosus and L. fermentum strains were able to coagulate milk.

the other hand, the second subgroup B (31% of L. plantarum

Coagulation of milk by some strains of L. plantarum and two

strains) showed a faster acidification rate. This subgroup

strains of L. rhamnosus revealed their potential as starters or

included L. plantarum strains showing a similar acidifying

adjunct cultures in production of fermented dairy food products

activity until 6 h but capable of achieving pH values ranging

(34).

between 1.05-1.13 and 1.96-2.14 pH units after 12 h and 24 h

Many strains of LAB produce exopolysaccharide (EPS),

of incubation.(iii) Fast acidifying L. plantarum strains (15% of

which might be a capsule, closely attached as slim (9). The

L. plantarum strains) showed the highest acidifying capacity.

production of EPS by Lactobacillus strains was examined in

This two L. plantarum strains, LbMS16 and LbMS21, showed

milk culture and on ruthenium red milk plates. Ruthenium red

a fast rate of acidifying ability until 6 h (0.73 and 0.92 pH units

stains the bacterial cell wall, thus producing red colonies for

respectively). This high capacity of acidifying milk by strains

nonropy strains. Production of EPS prevents this staining, and

of this cluster was also shown after 12 h and 24 h of

hence ropy colonies appear white on the same plates (44). The

incubation:

pH values were achieved, respectively, 1.31 to

results revealed EPS-production by isolated Lactobacillus

2.20 pH units for LbMS16 and 1.46 to 2.30 pH units for

strains (Table 4). Among the Lactobacillus strains tested, eight

LbMS21. Result of acidifying capacity of L. plantarum strains

showed EPS-production. Four L. plantarum (LbMS14,

after 6 h and 12 h of incubation is in agreement with several

LbMS16, LbMO16 and LbMO27 strains), two L. rharmnosus

authors (14, 23). In contrast, the acidifying rate of isolates after

(LbMF24 and LbMF25 strains), one L. pentosus (LbMT10

24 h of incubation was lower than those reported in the former

strain) and one L. fermentum (LbMA47 strain) produced EPS.

studies. The possibility to find groups of strains characterized

Exopolysaccharides play a major industrial role in the

by different acidifying ability is frequent in L. plantarum

production of fermented products, in particular for the

165

Marroki, A. et al.

Characterization of Lactobacillus and their technological properties

production of yoghurt, drinking yoghurt, cheese, fermented

Eleven percent of strains presented high activity to release Ala-

cream and milk-based desserts (18).

ρNA (13.12-14.10 U) and 16% of strains showed high ability

Results on aminopeptidase (AP) activity of 19 strains of

to release Leu-ρNA (10.10-11.37 U). Twenty six percent of

Lactobacillus strains tested using (Ala-ρNa and Leu-ρNa) are

tested strains presented medium Ala-aminopeptidase activity

shown in Figure 1. Amino acids released from peptides derived

(6.14-8.35 U) and 26% Leu-aminopeptidase (5.15-9.70 U).

from hydrolysis of casein may contribute directly or indirectly

However, 63% of isolates revealed low activity to degrade Ala-

for the development of flavour during ripening of cheese (48).

ρNA (1.02-4.93 U) and 58% to release Leu-ρNA (0.95-4.32

The proteolytic activity of dairy LAB is essential for the

U). All tested strains of L. pentosus, L. rhamnosus, and L.

bacterial growth in milk and it is involved in the development

fermentum exhibited low AP activity, except for L. pentosus

of sensory properties of different fermented milk products (11).

LbMT10 strain, which had medium Leu-aminopeptidase

Tested strains exhibiting aminopeptidase activity ranging

activity. Concerning the results obtained for L. plantarum

between 1.02 and 14.10 U for L-alanine-ρNA and 0.95 to

strains, two strains (LbMF13 and LbMO16) had high Ala-

11.37 U for L-leucine-ρNA. The AP was divided, according to

aminopeptidase activity and four strains (LbMS14, LbMS16,

the activity of each strain to high, medium and low activity.

LbMS24 and LbMO16) had high Leu-aminopeptidase activity.

Figure1. Aminopeptidase activity of strains of the Lactobacillus tested in this study.

Well diffusion assay was used to screen 19 Lactobacillus

932T) (Table 4). Bacteriocin like antimicrobial compound

strains isolated from goat’s milk for their antibacterial activities

production was indicated by a zone of clearing of more than 1

against several pathogenic indicator bacteria (Staphylococcus

mm against at least one of the indicator bacteria tested. The

T

aureus CECT 86 , Staphylococcus aureus UT 602, Bacillus

cell-free supernatants of five L. plantarum strains (38.46 %,

cereus INRA AVZ421 and Listeria monocytogenes CECT

LbMA9, LbMF13, LbMS14, LbMS24 and LbMO16) produced

166

Marroki, A. et al.

Characterization of Lactobacillus and their technological properties

an inhibition zone on agar against S. aureus CECT 86T and S.

according to Charteris et al. (10). All Lactobacillus strains

aureus UT 602 and three L. plantarum strains (23,07%,

isolated from goat’s milk were assayed for their susceptibility

LbMF13, LbMS24, and LbMO16) against Bacillus cereus

to eight antibiotics, using the disk diffusion method. Zone

AVZ 421. Antibacterial activity is a relatively frequent feature

diameters were measured, and strains were classified as,

of L. plantarum natural isolates (46). However, one strain of L.

susceptible (S), moderately susceptible (MS), and resistant (R)

rhamnosus strain (LbMF25) showed antimicrobial activity

(Table 4). No strains of Lactobacillus were totally susceptible

against S. aureus CECT 86T and S. aureus UT 602.

to all antibiotics tested and multiple resistances to most

Nevertheless, none of the tested strains of L. pentostus and L.

antibiotics were observed. Most strains were susceptible to -

fermentum displayed inhibitory activity against any of the

lactam, inhibitors of cell wall synthesis (penicillin G and

indicator bacteria. In addition, all strains tested were inactive

ampicillin). Some tested strains are moderately susceptible to

T

against Listeria monocytogenes CECT 932 . Complete

the former antibiotics. However, two L. plantarum strains,

inactivation of the antimicrobial activity was observed after

LbMO42 and LbMS24, were resistant to penicillin G or

treatment with proteinase K, indicating the proteinaceous

ampicillin, respectively, and one L. pentosus strain (LbMT9)

nature of the antimicrobial compound, whereas treatment with

was resistant to these two antibiotics simultaneously. Several

heat did not affect the inhibitory activity. Bacteriocins

studies

produced by LAB are of particular interest because of their

susceptibility to almost all penicillins (10). Nevertheless, the

potential use as natural food preservatives. The antibacterial

resistance of Lactobacillus strains to penicillin G and

activity potential of some L. plantarum strains against

ampicillin has been described in other studies (45, 13, 24).

Staphylococcus aureus (37, 6) and Bacillus cereus (6) has been previously reported.

report

that

species

of

lactobacilli

exhibited

Our result show that all tested strains were resistant to vancomycin, which is equally a cell wall synthesis inhibitor

Our results confirm the high incidence of bacteriocin-

(non- -lactam).

Resistance

of

Lactobacillus

species

to

producing lactic acid bacteria in milk samples, with inhibitory

vancomycin is due to the presence of D-Ala-D-lactate in their

activity against both pathogenic and spoilage microorganisms.

peptidoglycan, rather than the D-ala-D-ala dipeptide (28). Such

Goat’s milk may represent a source of new Lactobacillus

resistance is usually intrinsic, that is, chromosomally encoded

strains with the potential to inhibit undesirable and pathogenic

and nontransmissible (27). Concerning the protein synthesis

microorganisms for use in the biopreservation of dairy

inhibitors, all strains tested were susceptible to tetracyclin,

products. The resistance to high temperature, the proteinaceous

erythromycin and resistant to kanamycin and gentamycin.

nature, and the spectrum of activity of these antimicrobial

Lactobacilli are generally susceptible to antibiotics which

compound are advantageous for their use as biopreservatives in

inhibit the synthesis of proteins, such as erythromycin and

food (37).

tetracycline and more resistant to aminoglycosides (kanamycin and gentamicin) (10). Chloramphenicol inhibited most tested

Antibiotic resistance

strains. Three Lactobacillus strains showed a moderate

A key requirement for probiotic strains is that they should

susceptibility and one L. plantarum (LbMO42) strain was

not carry transferable antibiotic resistance genes. Transferable

resistant to this antibiotic. The high natural susceptibility of

resistance genes may pose a risk, as they can be transferred to

lactobacilli to chloramphenicol (protein synthesis inhibitor) is

pathogenic

well known (10, 45).

bacteria

(4).

In

this

study,

antibacterial

susceptibility testing of Lactobacillus strains was made

167

Marroki, A. et al.

Characterization of Lactobacillus and their technological properties

Table 4. Technological characteristics and antibiotic resistance of Lactobacillus strains isolated from Algerian goat’s milk pH Strains L. plantarum LbMA9 LbMF13 LbMF33 LbMS4 LbMS9 LbMS14 LbMS16 LbMS20 LbMS21 LbMS24 LbMO16 LbMO27 LbMO42 L. pentosus LbMS40 LbMT9 LbMT10 L. rhamnosus LbMF24 LbMF25 L. fermentum LbMA47

Coagulation EPS

Antibacterial activity

Antibiotics

S. aureus S. aureus CECT 86a UT 602b

Bacillus cereus Listeria INRA AVZ monocytogenes 421c CECT 932a

P

A

V

T

E

K

G

C

+ + + + -

+ + + + + -

+ + + + + -

+ + + -

-

MS S S S S S S MS S S S S R

S MS MS S S S S S S R MS S S

R R R R R R R R R R R R R

S S S S S S S S S S S S S

S S S S S S S S S S S S S

R R R R R R R R R R R R R

R R R R R R R R R R R R R

S S S S S S S S S S MS S R

-

+

-

-

-

-

S R S

S R MS

R R R

S S S

S S S

R R R

R R R

MS S S

1.87 1.96

+ +

+ +

+

+

-

-

S S

S S

R R

S S

S S

R R

R R

S S

1.35

-

+

-

-

-

-

S

MS

R

S

S

R

R

MS

6h

12h

24h

24h

0.27 0.50 0.52 0.53 0.51 0.56 0.73 0.57 0.92 0.56 0.27 0.56 0.40

1.01 0.97 0.78 1.05 1.13 1.11 1.31 1.07 1.46 0.91 0.89 0.98 0.86

1.60 1.46 1.16 2.14 1.97 2.07 2.20 1.96 2.30 1.49 1.43 1.74 1.33

+ + + + + + + -

0.43 0.56 0.53

0.89 1.19 0.76

1.29 1.3 1.18

0.63 0.69

1.10 1.40

0.25

0.83

+: Positive reaction; -: Negative reaction. EPS: Exopolysaccharide production (a) CECT: Colección Española de Cultivos Tipo, Valencia, Spain (b) U.T: University of Tlemcen laboratory collection, Algeria (c) INRA AVZ : Station de Technologie des Produits Végétaux, Institut national de la Recherche Agronomique, Avignon, France (INRA) PG: penicillin (10µg); A: ampicillin (10µg); V: vancomycin (30µg); T: tetracycline (30µg); E: erythromycin (15µg); K: kanamycin (30µg); G: gentamicin (10µg); C: chloramphenicol (30µg). S: sensitive strain; MS: moderately resistant strains; R: resistant strain.

168

Marroki, A. et al.

Characterization of Lactobacillus and their technological properties

With regard to antibiotic susceptibility profiles, L.

noted, is licensed under a Creative Commons License

plantarum strain (LbMO42) and L. pentosus strain (LbMT9)

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